Biomedical subjects

T Cowen

Publications and source records attributed to T Cowen.

At least 19 recordsLinked to original sources

PubMed2006

Reactive oxygen species, dietary restriction and neurotrophic factors in age-related loss of myenteric neurons.

We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.

Aging↗

PubMed2005

Vulnerability to ROS-induced cell death in ageing articular cartilage: the role of antioxidant enzyme activity.

OBJECTIVES: To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence. METHODS: Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured. RESULTS: Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered. CONCLUSIONS: Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.

Animals↗

PubMed2004

Neurodegeneration: a key factor in the ageing gut.

Many individuals experience gastrointestinal (GI) dysfunction more frequently as they age, and the segment of the human population that is growing the most rapidly is the 'oldest old', who are >/= 80 years old. There has recently been renewed interest in the age-related changes intrinsic to the gut, and these investigations may help physicians understand the 'normal' aged GI tract, as distinct from disordered bowel function that is the result of comorbid conditions and/or GI side effects of medications used to treat those conditions. In this concise review we summarize recent data that suggest age-related neurodegenerative changes in the enteric nervous system (ENS) are key to functional changes observed with advanced age. Morphological studies are reviewed that demonstrate clearly the loss of enteric neurones in both submucosal and myenteric plexuses in humans and in rodents. Recent studies that indicate selective preservation of nitrergic, but not cholinergic, neurones are reviewed, as are preliminary findings that intrinsic sensory neurones may be among the most 'age-labile' subpopulations of the ENS. Caloric restriction remains the only intervention known that prevents neurodegeneration of ageing in the ENS, and mechanisms involved in this phenomenon are discussed. The field of ageing research in enteric neurobiology is ripe for rapid progression from phenomenology of age-related losses of neurones and associated functional changes to discovery of therapeutic approaches that may help ameliorate deterioration of bowel function and thereby contribute significantly to improved quality of life in advanced age.

Aged↗

PubMed2003

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA.

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.

Adrenergic Agents↗

PubMed2003

Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat.

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.

Adrenergic Fibers↗

PubMed2002

The oestrogenized rat myometrium inhibits organotypic sympathetic reinnervation.

Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.

Animals↗

PubMed2002

Differential effects of oestrogen on developing and mature uterine sympathetic nerves.

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.

Animals↗

PubMed2002

Selective vulnerability in adult and ageing mammalian neurons.

Data are presented in support of the idea of antagonistic pleiotropy that features which are adaptive during early life may become maladaptive during the ageing process, when selective pressure is reduced. A model of selective vulnerability to age-related neurodegeneration involving neighbouring subpopulations of vulnerable and protected sympathetic neurons is presented. The two groups of neurons are morphologically and physiologically distinct, indicating advantageous adaptation to particular functions. Neurotrophin signalling is investigated in these different groups of neurons, revealing significant differences between them: neurotrophic factor expression in their target tissues is markedly different, same as their neurotrophin uptake characteristics. Preliminary evidence is presented that the mechanism linking neurotrophin signalling and age-related neurodegeneration may involve the capacity of neurons to buffer free radical generation, hence reducing the effects of attrition by free radical damage.

Aging↗

PubMed2001

p75 and TrkA receptors are both required for uptake of NGF in adult sympathetic neurons: use of a novel fluorescent NGF conjugate.

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red-nerve growth factor (DTR-NGF) conjugate. DTR-NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR-NGF labelling in SCG neurons was shown to be dose-dependent with an EC(50) of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR-NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85-93%) reduction in numbers of DTR-NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.

Animals↗

PubMed2001

Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons.

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

Amino Acid Chloromethyl Ketones↗

PubMed2001

The sweating apparatus in growth hormone deficiency, following treatment with r-hGH and in acromegaly.

Adult growth hormone deficient patients are known to exhibit reduced sweating and their ability to thermoregulate is diminished. Treatment of these patients with recombinant human growth hormone (r-hGH) is claimed to reverse these abnormalities. We have investigated this claim, as well as the mechanism underlying these altered sweating responses in GH-deficient patients as part of a placebo-controlled study on the effects of 6-12 months r-hGH therapy. Skin biopsies were obtained from these subjects and changes in morphology and innervation parameters for the eccrine sweat glands were examined. These included histochemistry for acetylcholinesterase (AChE) and immunohistochemistry for the neuropeptide vasoactive intestinal polypeptide (VIP) and for PGP9.5, a general neuronal marker. Sweat gland acinar size and periacinar innervation were measured by computerised image analysis. The patients underwent pilocarpine iontophoresis sweat rate tests and their serum insulin-like growth factor 1 (IGF-1) levels were assessed. Since active acromegaly involves excess GH secretion and hyperhidrosis, skin biopsies and sweat tests were also carried out on a group of these patients, as well as on control subjects. We have demonstrated a sweating defect in adult GH-deficiency which is accompanied by a reduction in AChE and VIP levels in the nerve supply to sweat glands. Following r-hGH therapy, an increase in AChE and VIP staining is seen in the sudomotor nerves accompanied by restoration of sweat rates and serum IGF-1 levels. Hence, normalization of sweat gland function includes recovery of sudomotor synapse constituents. A trophic effect of GH on sweat gland epithelium and/or on the associated nerves is proposed, supported by the observation that in acromegaly the size of sweat gland acini and the density of innervation to the sweat glands was greater than in controls.

Acetylcholinesterase↗

PubMed2001

Differential regulation of survival and growth in adult sympathetic neurons: an in vitro study of neurotrophin responsiveness.

The survival and growth of embryonic and postnatal sympathetic neurons is dependent on both NGF and NT3. While it has been established that adult sensory neurons survive independently of neurotrophins, the case is less clear for adult sympathetic neurons, where the studies of survival responses to neurotrophins have relied upon using long-term cultures of embryonic neurons. We have previously established a method to culture purified young (7 day) and adult (12 week) sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) in order to examine their survival and growth responses to neurotrophins. We now show that by 12 weeks after birth virtually all neurons (90%) survive for 24 h in the absence of neurotrophins. Neuron survival is unaffected by treatment with anti-NGF antibodies (anti-NGF) or with the tyrosine kinase inhibitor, K252a, confirming the lack of dependence on extrinsic neurotrophins. Duration of neuron survival in culture increases significantly between E19 and day 7 and week 12 posnatally, and is similarly unaffected by the presence of anti-NGF or K252a. Saturating concentrations of NGF and NT3 are equipotent in promoting neurite extension and branching. However, we find that NGF is more potent than NT3 in promoting neurite growth, irrespective of postnatal age. The growth-promoting effects of NGF and NT3 are almost entirely blocked by K252a, demonstrating that these effects are mediated via activation of Trk receptors, which therefore appear to remain crucial to plasticity of adult neurons. Our results indicate that maturing neurons acquire protection against cell death, induced in the absence of neurotrophin, while retaining their growth responsiveness to these factors.

Animals↗

PubMed2001

Serum-free culture of dissociated, purified adult and aged sympathetic neurons and quantitative assays of growth and survival.

In vitro studies of dissociated neurons have provided crucial data regarding the regulation of plasticity in embryonic and perinatal neurons from both central and peripheral nervous systems. There have been few attempts to apply these methods to adult or aged neurons and the methods that have been reported have not been able to dissect the possible confounding contributions of non-neuronal cells and serum. Furthermore, quantitative assays of cultured neurons, particularly of their growth, have rarely been described. We report here the development of a novel method for the dissociation, purification and culture of sympathetic neurons from the adult and aged rat SCG under serum free conditions and in defined media. The technique results in a relatively high yield of viable, growing neurons. We describe methods for assaying the total yield of neurons, the proportion of surviving neurons and the proportion of neurons initiating neurite outgrowth after plating. A novel semiautomated assay of neurite outgrowth is outlined using image analysis of composite images of immunofluorescence-stained single neurons.

Animals↗

PubMed2001

Innervation of cerebral blood vessels: morphology, plasticity, age-related, and Alzheimer's disease-related neurodegeneration.

The light microscopical and ultrastructural morphology of the innervation of the major cerebral arteries and pial vessels is described, including the origins of the different groups of nerve fibres and their characteristic neurotransmitter phenotype. Species and region specific variations are described and novel data regarding the parasympathetic innervation of cerebral vessels are presented. The dynamic nature, or plasticity, of cerebrovascular innervation is emphasized in describing changes affecting particular subpopulations of neurons during normal ageing and in Alzheimer's disease. The molecular controls on plasticity are discussed with particular reference to target-associated factors such as the neurotrophins and their neuronal receptors, as well as extracellular matrix related factors such as laminin. Hypotheses are presented regarding the principal extrinsic and intrinsic influences on plasticity of the cerebrovascular innervation.

Aging↗

PubMed2001

A heady message for lifespan regulation.

Mutant Caenorhabditis elegans in which the age-1 and daf-2 genes (involved in insulin-receptor-like signalling) are expressed at low levels exhibit extended lifespan. Wolkow and colleagues recently showed that restricted re-expression of age-1 and daf-2 genes in neurons of these mutants rescues wild-type lifespan as effectively as ubiquitous re-expression. Low levels of insulin-like signalling in neurons might control longevity by enhancing protection against free radical damage. However, in mammalian cells (including neurons) reduced insulin-like signalling is generally thought to be deleterious to antioxidant defence and to neuron survival. Here we discuss the new work and several hypotheses to explain this apparent contradiction.

Animals↗

PubMed2001

Cavernous sinus ganglia are sources for parasympathetic innervation of cerebral arteries in rat.

Retrograde tracing and immunohistochemistry was used in rats to investigate whether the ganglia in the cavernous sinus contribute to cerebrovascular innervation. The cavernous sinus ganglia in rat include the cavernous part of the pterygopalatine ganglion (PGC) and small cavernous ganglia (CG). The tracers, fluorogold and fast blue, were applied to the middle cerebral artery in eight rats. After 1 to 4 days, the cavernous sinuses were dissected out and studied as whole mount preparations and sections. A moderate number of labeled neurons were visible in the ipsilateral PGC and CG. Furthermore, fibers in the cavernous nerve plexus and abducens nerve were labeled, suggesting that the pathway from the cavernous sinus ganglia to the cerebral arteries runs through the cavernous plexus and then retrogradely along the abducens nerve to the internal carotid artery. Selected sections were immunohistochemically stained for the cholinergic marker, vesicular acetylcholine transporter (VAChT). Most cells in the PGC and CG were VAChT-immunoreactive, some of which also contained tracer. It is concluded that in rat, the cavernous sinus ganglia, consisting of the PGC and small CG, contribute to parasympathetic cerebrovascular innervation and that the cavernous nerve plexus and abducens nerve are involved in the pathway from these ganglia to the cerebral arteries.

Acetylcholinesterase↗

PubMed2000

The role of NGF in pregnancy-induced degeneration and regeneration of sympathetic nerves in the guinea pig uterus.

In the guinea pig, pregnancy is associated with a generalised depletion of noradrenaline in uterine sympathetic nerves and, in the areas of the uterus surrounding the foetus, by a complete degeneration of sympathetic nerve fibres. These pregnancy-induced changes have been interpreted as a selective effect of placental hormones on the system of short sympathetic fibres arising from the paracervical ganglia. An alternative explanation is that pregnancy affects the neurotrophic capacity of the uterus. We measured NGF-protein levels in the guinea pig uterine horn, tubal end and cervix at early pregnancy, late pregnancy and early postpartum, using a two-site enzyme-linked immunosorbent assay. For comparative purposes the distribution and relative density of noradrenaline-containing sympathetic nerve fibres were assessed histochemically, and tissue levels of noradrenaline were measured biochemically, using high-performance liquid chromatography with electrochemical detection. In all the uterine regions analysed, NGF-protein levels showed a decline at term pregnancy, but in no case was this change statistically significant. After delivery, NGF-protein levels showed a marked increase in the cervix as well as in both the fertile and empty horns. These results suggest that alterations in NGF-protein do not account for the impairment of uterine sympathetic innervation during pregnancy, but may contribute to their recovery after delivery.

Animals↗

PubMed2000

Symposium on mechanisms of ageing and longevity.

The Symposium on 'Mechanisms of Ageing and Longevity' was held at the Royal Free Campus, Royal Free and University College Medical School, London, on 17 September 1999. It was organised and sponsored by the Anatomical Society of Great Britain and Northern Ireland with generous contributions from BBSRC, AgeNet and Research into Ageing.THE SYMPOSIUM WAS ORGANISED AS A RESULT OF RECENT DISCOVERIES IN TWO PRINCIPAL AREAS: the genetics of longevity regulation in the nematode worm, Caenorhabditis elegans, and the cell biology of ageing in mammalian cells. Papers were presented covering the role of insulin-like signalling, free radical damage and caloric restriction in ageing of invertebrate and mammalian systems. One of the principal aims was to examine the idea that a genetically conserved signalling pathway might regulate ageing processes at both a cellular and an organismal level.

Journal Article↗